橡胶延伸因子基因及3′端非编码区的克隆与序列分析

橡胶延伸因子ＲＥＦ（Ｒｕｂｂｅｒ Ｅｌｏｎｇａｔｉｏｎ Ｆａｃｔｏｒ）是橡胶生物合成中最重要的酶之一,作者利用ＲＥＦ基因序列设计并合成引物。通过提取胶乳总ＲＮＡ用ＲＴ法合成ｃＤＮＡ后,通过ＰＣＲ技术坟民并克隆了ＲＥＦ基因和３′端非编码区,序列测定结果表明,ＲＥＦ基因全长４１４个碱基,编码１３８个氨基酸,３′端非编码区长１１２ｂｐ．与ＧｏｙｖａｅｒｔｓＥ等人发表的序列比较：ＲＥＦ基因同源率为１００％     

橡胶延伸因子基因及3'端非编码区的克隆与序列分析

橡胶延伸因子ＲＥＦ（Ｒｕｂｂｅｒ Ｅｌｏｎｇａｔｉｏｎ Ｆａｃｔｏｒ）是橡胶生物合成中最重要的酶之一．作者利用ＲＥＦ基因序列设计并合成引物． 通过提取胶乳总ＲＮＡ 用ＲＴ法合成ｃＤＮＡ 后,通过ＰＣＲ技术扩增并克隆了ＲＥＦ基因和３端非编码区． 序列测定结果表明,ＲＥＦ基因全长４１４ 个碱基,编码１３８ 个氨基酸,３端非编码区长１１２ ｂｐ． 与Ｇｏｙｖａｅｒｔｓ Ｅ 等人发表的序列比较：ＲＥＦ基因同源率为１００％ ,３端非编码区有２ ｂｐ 的差异,同源率为９８％ ． 将所克隆的ＲＥＦ基因及３非编码区进行地高辛标记, 对胶乳和叶片总ＲＮＡ 进行点杂交分析,结果表明,胶乳总ＲＮＡ 呈阳性反应,叶片总ＲＮＡ 则呈阴性反应．     

文摘

010 0 51炭疽杆菌水肿因子基因的克隆与序列测定 [中 ]/袁斌… / /生物技术通讯 .- 2 0 0 0 ,11( 4 ) .- 2 4 9～ 2 51采用聚合酶链反应 (ＰＣＲ)从炭疽芽孢杆菌减毒株Ｙｂ1中扩增其水肿因子 (ＥＦ)的编码区基因 ,将其克隆至ｐＧＥＭ -Ｔ载体中 ,并分步测定其序列。序列测定表明 ,该基因长 2 30 1ｂｐ ,编码 2 67个氨基酸 ,与已报道的Ｓｔｅｒｎｅ标准株的ＥＦ基因完全一致。 (紫玉 )0 10 0 52人骨形态发生蛋白 - 7全长ｃＤＮＡ的克隆及序列测定 [中 ]/饶亚兰… / /生物技术通讯 .- 2 0 0 0 ,11( 4 ).- 2 52～ 2 53从人胎儿肾中提取总ＲＮＡ…     

紫云英根瘤菌胞外多糖合成基因exol的序列分析

对互补紫云英根瘤菌ＥｘｏＮｄｖＦｉｘ变种的２．６ｋｂＤＮＡ片段进行序列测定,分析结果表明,该片段内存在的一个完整的阅读框架（ＯＲＦ）ｅｘｏｌ。ｅｘｏｌ编码了３４０个氨基酸,为细胞质蛋白。Ｅｘｏｌ蛋白序列与苜蓿根瘤菌的糖基转移酶ＥｘｏＵ有较强的同源性,利用启动子检测质粒,分析ｅｘｏｌ基因的表达,发现在ｅｘｏｌ基因上有两个启动子活性区段,Ｐｅｘｏｌａ位于基因前端,Ｐｅｘｏ１ｂ位于基因中间,Ｐｅｘｏｌａ     

炭疽杆菌保护性抗原基因的克隆与序列测定

采用聚合酶链反应从炭疽芽孢杆菌减毒株ＹＢ１中扩增其保护性抗原（ＰＡ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２２０５ｂｐ,编码７３５个氨基酸残基,与献报道的标准菌株Ｓｔｅｒｎｅ株的ＰＡ序列只有４个碱基的差异。     

抗甜菜坏死黄脉病毒单链抗体表达载体的构建及其表达

用ＰＣＲ方法以分泌抗甜菜坏死黄脉病毒（ＢＮＹＶＶ）单克隆抗体的杂交瘤细胞的基因组为模板,扩增了编码ＢＮＹＶＶ单抗的重链可变区（ＶＨ）基因。测序表明,该ＶＨ序列属于小鼠ＩＩ（Ａ）亚类,全长为３６０ｂｐ,编码１２０个氨基酸。将其和先前克隆的轻链基因分别插入到一个含有连接ＶＨ和ＶＬ基因的连接序列的质粒之中,构建成单链抗体（ｓｃＦｖ）基因的表达载体ｐＴＣｓｃＦｖ。将质粒在大肠杆菌中表达,ＥＬＩＳＡ法检测出     

紫云英根瘤菌胞外多糖合成基因exo1的序列分析

对互补紫云英根瘤菌Ｅｘｏ－Ｎｄｖ－Ｆｉｘ－变种的２．６ｋｂＤＮＡ片段进行序列测定。分析结果表明,该片段内存在一个完整的阅读框架（ＯＲＦ）ｅｘｏ１。ｅｘｏ１编码３４０个氨基酸,为细胞质蛋白。Ｅｘｏ１蛋白序列与苜蓿根瘤菌的糖基转移酶ＥｘｏＵ有较强的同源性。利用启动子检测质粒,分析ｅｘｏ１基因的表达,发现在ｅｘｏ１基因上有两个启动子活性区段,Ｐｅｘｏ１ａ位于基因前端,Ｐｅｘｏ１ｂ位于基因中间。Ｐｅｘｏ１ａ很可能包含了ｅｘｏ１基因的启动子。     

新城疫病毒F48E8株融合蛋白基因序列分析

本研究报道了新城疫病毒（ＮＤＶ）中国标准强毒株Ｆ４８Ｅ８融合蛋白（Ｆ）基因的序列。该基因核苷酸序列长度为１７００ｂｐ,编码由５５３个氨基酸组成的Ｆ０多肽。Ｆ０酶切激活部位序列为ＲＲＱＲＲ↓Ｆ,具有ＮＤＶ强毒的特征。Ｆ０中有３个主要由疏水性氨基酸组成的区域和６个可糖基化位点。经比较,ＮＤＶＦ４８Ｅ８株和Ｍｉｙａｄｅｒａ株、ＴｅｘａｓＧＢ株的氨基酸同源性分别为９３．６４％和９２．４１％。     

番茄花叶病毒外壳蛋白基因及3′端非编码区的克隆、序列分析及其表达

根据已报道的番茄花叶病毒Ｌ株系（ＴｏＭＶＬ）序列人工合成引物,经ＲＴＰＣＲ扩增并克隆了我国番茄花叶病毒分离物（ＴｏＭＶＳ１）的外壳蛋白ＣＰ基因及３′端非编码区。序列测定结果表明,所得ｃＤＮＡ共长６８２个核苷酸,其中ＣＰ基因含４８０个核苷酸,编码１５８个氨基酸,３′端非编码区含２０２个核苷酸,其核苷酸序列与ＴｏＭＶＬ株系具有９９．５％的同源率。将该基因片段克隆到ｐＧＥＭＥＸ１载体中,转入Ｅ．ｃｏｌｉ后诱导表达,经Ｗｅｓｔｅｒｎｂｌｏｔ检测证明,该基因已在大肠杆菌中正确表达。这是我国首次报道ＴｏＭＶＣＰ基因序列。     

神经元限制性沉默因子研究进展

神经元限制性沉默因子（ＮＲＳＦ）是神经元发育中的一个负调探因子。它与靶序列神经元限制性沉默子（ＮＲＳＥ）相互作用,主要调控神经元特异性基因,防止神经元基因在非神经元组织中的异位表达以及防止神经发生中神经元表型的提前表达。最近的研究表明,ＮＲＳＦ还可调控神经元转录编码基因和某些非神经元基因,暗示它在胚胎发育中可能起着更为广泛的作用。     

Functional EF-Tu with large C-terminal extensions in an E coli strain with a precise deletion of both chromosomal tuf genes

An Escherichia coli strain was constructed in which both chromosomal genes encoding elongation factor (EF)-Tu (tufA and tufB) have been inactivated with precise coding sequence replacements. A tufA gene in an expression vector is supplied as the sole EF-Tu source. By using plasmid replacement, based on plasmid incompatibility, mutant EF-Tu variants with a large C'-terminal extension up to 270 amino acids were studied and proved to be functional in a strain lacking the chromosomal tufA and tufB genes.     

结核分枝杆菌EF4敲除菌株的构建

EF4是一个由 lepA 基因编码的与蛋白质翻译密切相关的延伸因子,在细菌中高度保守,但其确切功能和分子机制尚不清楚,在结核分枝杆菌中的功能至今未见报道。为探索EF4在结核分枝杆菌中的功能,需构建一株结核分枝杆菌 lepA 基因敲除株。本研究以结核分枝杆菌H37Ra全基因组DNA为模板,设计并通过聚合酶链反应(polymerase chain reaction,PCR)扩增 lepA 基因左、右臂,连接到p0004S质粒,构建同源重组质粒p0004S-Δ lepA 。然后,通过噬菌体体外包装,将p0004S-Δ lepA 质粒连接到phAE159质粒,构建phAE159-Δ lepA 噬菌体包装质粒。在耻垢分枝杆菌mc 2155中大量扩增噬菌体并受结核分枝杆菌侵染进行同源重组,筛选阳性克隆,从基因组和蛋白质表达水平检测该突变株中 lepA 基因及EF4蛋白表达。PCR结果显示,敲除株基因组中 lepA 基因已被潮霉素抗性基因成功替换,蛋白免疫印迹结果显示该敲除株中无EF4表达,表明其为成功构建的Ra Δ lepA 。生长曲线分析显示,正常培养条件下,结核分枝杆菌野生株与敲除株生长趋势一致。敲除株与野生株在菌落形态上有一定差异,相比于野生株,Ra Δ lepA 菌落颜色发黄,凸起偏厚,生长过程中生物膜皱褶较少。耐胁迫能力分析显示,与野生株相比,Ra Δ lepA 耐热、抗去垢剂、抗氧化能力无显著差异,但耐酸性环境能力明显增强。本研究利用噬菌体介导的重组法成功构建了结核分枝杆菌 lepA 基因敲除株,为后续研究结核分枝杆菌EF4的功能提供了重要基础。     

Increased baculovirus susceptibility of armyworm larvae feeding on transgenic rice plants expressing an entomopoxvirus gene.

We have introduced an entomopoxvirus gene encoding a virus enhancing factor (EF) into rice, which resulted in high-level accumulation of the EF in the transgenic plants. The introduced gene was stably inherited in the progeny of the primary transformants, as shown by analysis of their genomic DNA. Bioassays for insect susceptibility to baculovirus infection showed that armyworm larvae feeding on the transgenic rice had increased susceptibility to a Nucleopolyhedrovirus. Thus, introduction of the EF gene into plants can be used as a strategy to increase the effectiveness of baculoviruses in insect pest management.     

A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1?, pXO2?), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1? A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture.     

The mosaic cox1 gene in the mitochondrial genome of Schizosaccharomyces pombe: minimal structural requirements and evolution of group I introns

The gene encoding subunit 1 of cytochrome oxidase (cox1) in the fission yeast Schizosaccharomyces pombe is polymorphic. In strain 50 it contains two group I introns with open reading frames (ORFs) in phase with the upstream exons (Lang, 1984). In strain EF1 two additional very short group I introns which do not possess ORFs were detected by DNA sequencing. These two introns (AI2a and AI3) share distinct characteristics concerning their nucleotide sequence and secondary structure and are located at identical positions as the introns AI4 and AI5 beta, respectively, in the cox1 gene of Saccharomyces cerevisiae. The sequence homology of the cob and cox1 genes around the splice points of introns AI2a, AI4, and BI4 (cob intron 4) might reflect horizontal gene transfer between the distantly related species S. pombe and S. cerevisiae.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. 3. Gene mapping in strain EF1 (CBS 356) and analysis of hybrids between the strains EF1 and ade7-50h-

The Schizosaccharomyces pombe strain EF1 (CBS 356) is haploid, prototrophic, respiratory competent, and of homothallic mating type. From restriction enzyme analysis the length of the mitochondrial genome is 17.3 kilobase pairs, which is in good agreement with the value of 17.1 kilobase pairs determined by electron microscopy. The mitochondrial genome of strain EF1 is thus about 2.3 kilobase pairs shorter than that of strain ade7-50h- (about 19.4 kilobase pairs). A restriction map was constructed for 11 enzymes: For most, but not all of them, the pattern is nearly identical to that of strain ade7-50h-. The genes for the large ribosomal RNA, the subunits 1, 2, and 3 of cytochrome c oxidase, subunits 6 and 9 of ATP synthetase, and cytochrome b were localized by hybridization with mitochondrial DNA probes from Saccharomyces cerevisiae. The gene order was found to be the same in both yeast strains. From Southern hybridization of strain ade7-50h- with nick-translated mitochondrial DNA from strain EF1 it is evident that strain EF1 does not possess the intron, which is present in the cytochrome b gene of Schizosaccharomyces pombe strain ade7-50h-. Crosses between strain ade7-50h- and EF1 demonstrate that both the nuclear and the mitochondrial genomes are able to recombine. The mitochondrial genomes of 2 out of 30 independently isolated hybrids between the two strains are described as the result of recombination between the two parental mitochondrial genomes.     

Construction of a non-antibiotic expression system in a marine cyanobacterium Synechococcus sp. PCC 7002 and its application in production of oral vaccine against enterotoxin of Escherichia coli

Synechococcus sp. PCC 7002 is a marine cyanobacterium that is rich in thylakoid membranes and performs oxygenic photosynthesis. In this study, we inactivated its psbEF genes encoding cytochrome b-559 of photosystem II and the mutant strain EF211 could not grow photoautotrophically. A non-antibiotic plasmid (pAQEF559) was constructed in such a way that it has a psbEF operon and all antibiotic genes are deleted. The plasmid could complement EF211 and restore photoautotrophical growth. This non-antibiotic plasmid was used to express the gene encoding heat-labile enterotoxin B-subunit, LT-B. The overproduced LT-B in the cyanobacterium could form pentamers and was associated with thylakoid membranes. Oral immunization of mice with the cyanobacterium overproducing LT-B showed that it was very effective in stimulating production of both IgG in serum and IgA in intestine. Our results demonstrate that the non-antibiotic vector in combination with the constructed host cyanobacterium is useful in production of recombinant proteins in cyanobacteria and the cyanobacterial cells producing LT-B can be an effective oral vaccine against enterotoxins.     

A novel recombinant ethyl ferulate esterase from Burkholderia multivorans

AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND RESULTS: A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column. CONCLUSION: Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF). SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.